The goal of this proposal is to understand the molecular basis of phase variation of the pyelonephritis-associated pili (pap) operon of uropathogenic Escherichia coli. The pap operon codes for a fimbrial-adhesin complex important in the pathogenesis of upper urinary tract infections. Pap phase variation is controlled by leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). These proteins act within the pap regulatory region, which includes two Dam target sites designated GATC-I and GATC-II. Competitive interactions between Lrp and Dam at GATC-I and GATC-II result in pap DNA methylation patterns characteristic of phase ON and phase OFF cells. These methylation patterns, in turn, control Lrp binding to pap regulatory DNA. A key step in the phase OFF to ON switch is the movement of Lrp from the GATC-II region to GATC-I, located over 100 basepairs upstream. This Lrp translocation is facilitated by PapI, which specifically binds to the Lrp moiety of Lrp-pap DNA complexes. One aim of this proposal is to identify the domains of PapI and Lrp that interact with each other to aid Lrp translocation. Both genetic and biochemical approaches will be used to identify the amino acids of Lrp and PapI that contact each other. A second aim is to determine how PapI and Dam affect the binding equilibria and stoichiometry of Lrp to pap DNA using gel shift, DNA footprinting, and fluorescence resonance energy transfer binding assays. A third aim is to isolate a recently identified factor that interacts with the pap regulatory region designated as Dam blocking factor (Dbf). Dbf will be purified by DNA affinity chromatography, and its role in regulating Pap phase variation will be assessed using a dbf null mutation for genetic analysis. A fourth aim is to determine the roles of Lrp, PapI, Dam, Dbf, and CAP in the activation and repression of pap transcription. In vitro transcription experiments will be carried out to measure the effects of these regulatory factors on the affinity of RNA polymerase for the papBAp promoter, the rate of open complex formation, and the rate of promoter clearance. The relevance of the in vitro data to Pap gene regulation in E. coli will be determined using in vivo footprinting. The studies proposed should provide a solid basis for understanding how DNA methylation patterns are formed and regulated by protein-DNA interactions and how methylation patterns, in turn, control gene expression.